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Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor with an essential role in cytoskeletal rearrangement, cell migration, and survival of various immune cells. Interestingly, DOCK8-deficient mice are resistant to the development of experimental autoimmune encephalomyelitis (EAE). To understand if EAE resistance in these mice results from an alteration in dendritic cell (DC) functions, we generated mice with conditional deletion of DOCK8 in DCs and observed attenuated EAE in these mice compared with control mice. Additionally, we demonstrated that DOCK8 is important for the existence of splenic conventional DC2 and lymph node migratory DCs and further established that migratory DC, rather than resident DC, are essential for the generation and proliferation of pathogenic T cell populations upon immunization with myelin Ag in adjuvant. Therefore, our data suggest that limiting migratory DCs through DOCK8 deletion and possibly other mechanisms could limit the development of CNS autoimmunity.

Thymic regulatory T cells (tTregs) are potent inhibitors of autoreactive immune responses, and loss of tTreg function results in fatal autoimmune disease. Defects in tTreg number or function are also implicated in multiple autoimmune diseases, leading to growing interest in use of Treg as cell therapies to establish immune tolerance. Because tTregs are present at low numbers in circulating blood and may be challenging to purify and expand and also inherently defective in some subjects, we designed an alternative strategy to create autologous Treg-like cells from bulk CD4+ T cells. We used homology-directed repair (HDR)-based gene editing to enforce expression of FOXP3, the master transcription factor for tTreg Targeted insertion of a robust enhancer/promoter proximal to the first coding exon bypassed epigenetic silencing, permitting stable and robust expression of endogenous FOXP3. HDR-edited T cells, edTregs, manifested a transcriptional program leading to sustained expression of canonical markers and suppressive activity of tTreg Both human and murine edTregs mediated immunosuppression in vivo in models of inflammatory disease. Further, this engineering strategy permitted generation of antigen-specific edTreg with robust in vitro and in vivo functional activity. Last, edTreg could be enriched and expanded at scale using clinically relevant methods. Together, these findings suggest that edTreg production may permit broad future clinical application.

Foamy viruses (FV) are ideal vectors for in vivo gene therapy because FV infection is non-pathogenic in people, FV are more resistant to serum inactivation than lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and FV exhibit favorable proviral integration patterns compared to gammaretroviruses. In vivo gene therapy with a FV vector expressing the human common gamma chain (γC) under the short EF1α promoter results in a functional immune reconstitution in X-SCID dogs.

In both humans and canines, X-linked severe combined immunodeficiency disease (XSCID) is caused by mutations in the interleukin-2 receptor gamma chain gene (IL2RG) which results in a lack of response to common gamma-chain (gammaC) dependent cytokines and abnormal development of T and B lymphocytes, and natural killer (NK) cells. Death from infections usually occurs before 1 year of age unless allogeneic hematopoietic cell transplantation (HCT) is performed. While HCT is successful if an HLA-matched sibling donor is available, transplants from mismatched and unrelated donors are associated with greater morbidity and overall survival can be as low as 50%. To circumvent these complications, several clinical trials are testing the possibility of utilizing blood and marrow stem cells from the patient for ex vivo gene therapy to treat X-SCID. Although these trials show promising results, they require expensive GMP cell manufacturing that are not accessible to many patients, and may also necessitate low level of conditioning to improve engraftment of gene-corrected cells. With these limitations in mind, we have explored in vivo gene therapy as a treatment for X-SCID. We previously showed that foamy virus vectors (FVs), exhibit a potentially more favorable integration profile compared to lenti- and gamma-retroviral vectors. In vivo delivery of a gammaC-FV in dogs resulted in immune reconstitution with gene-corrected T cells in dogs but the treated animals still developed infections and had low levels of immunoglobulin levels. We hypothesized that an increased transduction of hematopoietic stem/progenitor cells in vivo might result in more rapid and sustained immune reconstitution. Thus, in the current study, we used cG-CSF and AMD3100 to mobilize hematopoietic stem/progenitor cells into the peripheral blood prior to in vivo injection with a FV expressing the gammaC gene driven by a PGK promoter (PGK-gammaC-FV). We mobilized two X-SCID dogs at ~3 weeks of age with 5ug/kg of cG-CSF bi-daily from day -4 to -1 prior to FV injection, and with 4mg/kg of AMD3100 on the morning of the injection with 4x10e8 IU of PGK-gammaC-FV. Our mobilization protocol resulted in a 10-fold increase in CD34+ cells in the peripheral blood of mobilized X-SCID dogs as compared to a unmobilized normal littermate control (Figure 1 A). Lymphocyte recovery and gene marking in the mobilized animals was significantly improved as compared to animals that were previously injected with similar doses of either PGK-gammaC-FV or EF1a-gammaC-FV but without mobilization. As illustrated in Figure 1B-C, lymphocyte counts expanded to ~3000 cells/uL with ~75% gene marking in the mobilized animals treated with PGK-gammC-FV within 30 days, as compared to Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Scharenberg:bluebird bio: Consultancy, Equity Ownership, Research Funding; Alpine Immune Sciences: Consultancy. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

This study reports the identification of a novel heterozygous IKBA missense mutation (p.M37K) in a boy presenting with ectodermal dysplasia with immunodeficiency (EDA-ID) who had wild type IKBKG gene encoding NEMO. Our aim was to characterize the clinical course of this IκB-α gain-of-function mutant and to investigate if the p.M37K substitution affects NF-κB activation by interfering with IκB-α degradation, thus impairing NF-κB signaling and causing the EDA-ID phenotype.NF-κB signaling was evaluated by measuring IκB-α degradation in patient fibroblasts. In addition, transiently transfected HeLa cells expressing either the M37K-mutant IκB-α allele, the previously characterized S36A-mutant IκB-α allele, or wild type IκB-α were evaluated for IκB-α degradation and NF-κB nuclear translocation following stimulation with TNF-α.Clinical findings revealed a classical ectodermal dysplasia phenotype complicated by recurrent mucocutaneous candidiasis, hypothyroidism, hypopituitarism, and profound combined immunodeficiency with decreased numbers of IL-17 T cells. IκB-α degradation after TNF-α and TLR agonist stimulation was abolished in patient fibroblasts as well as in HeLa cells expressing M37K-IκB-α similar to cells expressing S36A-IκB-α resulting in impaired nuclear translocation of NF-κB and reduced NF-κB dependent luciferase activity compared to cells expressing wild type IκB-α. Patient whole blood cells failed to secrete IL-6 in response to IL-1ß, Pam2CSK4, showed reduced responses to LPS and PMA/Ionomycin, and lacked IL-10 production in response to TNF-α.The novel heterozygous mutation p.M37K in IκB-α impairs NF-κB activation causing autosomal dominant EDA-ID with an expanded clinical phenotype.

In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy because they are non-pathogenic in humans, they exhibit increased serum stability and they integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human common gamma chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of functional CD3+ lymphocytes in canine X-SCID (Burtner CR et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood. 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy that include different promoters and different fluorophores. We performed a head-to-head comparison of two promoters, our previously used EF1α promoter and the human phosphoglycerate kinase (PGK) promoter, by simultaneously injecting three X-SCID pups with equal titers of two therapeutic, human γC-encoding FV vectors. These vectors expressed the fluorophores GFP or mCherry to allow for tracking of transduced cells. Each dog received between 3 and 4 x 108 infectious units of each FV vector. In all treated dogs, lymphocyte marking in the PGK arm reached 50% between day 60 and day 110 post-injection and continued to expand over time, while the EF1α arm peaked at day 42 and never expanded above 10% (Fig 1A). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the EF1α arm, suggesting competition within the expanding T cell niche. The development of total CD3+ T cells achieved therapeutic levels (1000 cells/μL of blood) in all three dogs between day 70 and day 130 post-treatment (Fig 1B). We further validated the functionality of these cells by showing that they express a diverse T cell receptor repertoire using spectratyping analysis. In addition, peripheral blood mononuclear cells from the treated animals could be activated in vitro by exposure to the mitogen Phytohemagglutinin A at a level comparable to normal cells. Immunization of the treated dogs with bacteriophage ΦX174 showed production of specific IgG antibodies, suggesting the ability of B lymphocytes to undergo isotype switching. Finally, retroviral integration site analysis revealed polyclonal contribution to the reconstituting T cells. In summary, our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. The outcome in dogs was substantially improved compared to our previous study using EF1α, where robust lymphocyte marking was achieved in only 2 of 5 dogs, and where clonal dominance was observed. Ongoing work will determine whether the superior performance of the PGK vector is due to higher γC expression in PGK vs. EF1α corrected cells. Figure 1. T-cells expansion in X-SCID dogs following FV treatment. A) Percent of gene-modified peripheral blood lymphocytes in each experimental arm after in vivo gene therapy. B) Absolute CD3+ count per μL peripheral blood in all treated animals. Dotted line indicates therapeutic counts of CD3+ cells. Figure 1. T-cells expansion in X-SCID dogs following FV treatment. A) Percent of gene-modified peripheral blood lymphocytes in each experimental arm after in vivo gene therapy. B) Absolute CD3+ count per μL peripheral blood in all treated animals. Dotted line indicates therapeutic counts of CD3+ cells. Disclosures No relevant conflicts of interest to declare.

In vivo gene therapy has several benefits over ex vivo hematopoietic stem cell gene therapy, including the correction of progenitor cells in their native environments, the portability of the treatment to the patient, and the ability to administer serial doses of therapeutic vector. Foamy viruses (FV) are ideal vectors for in vivo gene therapy for 3 primary reasons: (1) FV are non-pathogenic in humans, (2) they exhibit enhanced serum stability as compared to lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and (3) FV integrate into host genomes with a favorable integration pattern. We recently demonstrated that intravenous injection of a FV vector expressing the human common gamma chain (γC) under the constitutively active short elongation factor 1α (EF1α) promoter is sufficient to drive development of CD3+ lymphocytes in canine X-SCID, which undergo T cell receptor rearrangement and exhibit a functional signaling response to T cell activating mitogens (Burtner CR, Beard BC, Kennedy DR, et al. Intravenous injection of a foamy virus vector to correct canine SCID-X1. Blood. 2014;123(23):3578-84). However, retroviral integration site analysis in that study indicated that T cell reconstitution occurred through the correction of a limited number of progenitors, possibly due to sub-therapeutic expression levels from the EF1α promoter. To address this issue, we are evaluating multiple parameters of vector design for in vivo gene therapy, including different promoters, using injections of vectors marked with different fluorophores. Preliminary data indicated that ex vivo transduction of canine CD34+ cells with a FV vector expressing human γC and a fluorescent reporter under the human phosphoglycerate kinase (PGK) promoter resulted in higher transduction efficiencies and increased mean fluorescence intensity, compared to that of an identical vector containing the EF1α promoter. We therefore performed a head-to-head comparison of the two promoters by simultaneously injecting X-SCID pups with equal titers of 2 therapeutic, human γC-encoding FV vectors that differed only in the promoter used to drive human γC expression and in the fluorophore color to distinguish gene-marked cells (GFP and mCherry). Each dog received 4 x 108 infectious units of each FV vector. A significant population of gene-marked lymphocytes appeared in the PGK arm 42 days post in vivo gene therapy, which continued to expand over the next two months of follow-up (Fig 1A). By 84 days post injection, lymphocyte gene marking in the competitive PGK arm reached 60% in both dogs. For comparison, this robust level of lymphocyte gene marking was achieved in only 2 of 5 dogs after 122 and 160 days, respectively, in our previous EF1α virus treated cohort. In contrast, the EF1α arm peaked at 42 days after in vivo gene therapy and never expanded above 10% (Fig 1A). Interestingly, the expansion of T lymphocytes from gene-modified cells expressing γC under the PGK promoter appeared to preclude further development of T cells by the by the EF1α arm, suggesting competition within the expanding T cell niche. The expansion of gene-marked lymphocytes was followed by the development of CD3+ T cells, leading to a therapeutic level of CD3+ cells (1000 cells/μl of blood) in both dogs (Fig 1B). Additionally, our data indicate low but persistent gene marking in other blood cells, including granulocytes and B cells, with B cell marking in one animal exceeding 2% in the PGK arm. Our data suggest that the PGK promoter results in a robust and sustained correction of progenitor T cells in a relevant large-animal disease model for primary immunodeficiency. These data also highlight the utility of the in vivo approach to explore key parameters of vector design in competitive repopulation experiments that may be useful for other diseases. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.